Methods for Identifying Agents that Inhibit Cell Migration, Promote Cell Adhesion and Prevent Metastasis

ABSTRACT

Disclosed are methods for identification of agents that modulate cell attachment, cell migration and cell viability. Cancer and primary cells adhered to a matrix are treated with agent(s) that modulate ActRII signaling and cell adhesion. Agents are tested that modulate cell adhesion, detachment, invasion and viability. Agents that modulate the expression, phosphorylation, function and translocation of ActRII signaling pathway members also can predict agents that modulate cell adhesion, detachment, invasion and viability. The methods have utility in identifying agents that prevent cancer cell metastasis, wound dehiscence, aortic dissection and aid retina attachment and skin replacement and fertility.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional U.S. patent applicationfiled Feb. 20, 2009, having a Ser. No. 61/154,251, the disclosure ofwhich is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to an assay that can identify agents that inhibitcell migration, promote cell adhesion and prevent metastasis.

BACKGROUND OF THE INVENTION

Metastasis of cells from a primary tumor in mammals leads to the spreadof cancer to other tissues of the body (1-3). The spread of metastasesmay occur via the blood or the lymphatics or through both routes. Cancercell metastasis commonly occur in lungs, liver, brain, and the bones.These secondary aggressive cancers lead to organ dysfunction andultimately death (Metastatic Cancer: Questions and Answers”. NationalCancer Institute). To date, there are few effective treatments formetastatic cancers.

Signaling pathways that promote cell detachment and/or metastasis arelargely unknown. However, we have discovered signaling pathways thatunexpectedly regulate cell adhesion to the extra-cellular matrix (ECM)are prime candidates for promoting metastasis (4). These pathways may beimpacted upon by the many genetic and epigenetic changes as a celltransitions to malignancy, together with microenvironmental changes suchas changes in hormone signaling.

Wound dehiscence is the premature “bursting” open of a wound alongsurgical suture. It is a surgical complication that results from poorwound healing. Risk factors are age, diabetes, obesity, poorknotting/grabbing of stitches and trauma to the wound after surgery.Agents that promote cell adhesion could speed healing. Retinaldetachment is a disorder of the eye in which the retina peels away fromits underlying layer of support tissue. Initial detachment may belocalized, but without rapid treatment the entire retina may detach,leading to vision loss and blindness. Agents that promote cell adhesioncould speed healing. Aortic dissection is a tear in the wall of theaorta that causes blood to flow between the layers of the wall of theaorta and force the layers apart. Aortic dissection is a medicalemergency and can quickly lead to death, even with optimal treatment. Ifthe dissection tears the aorta completely open (through all threelayers), massive and rapid blood loss occurs. Agents that promote celladhesion could speed healing. Major skin trauma (e.g. burns, amputation)can be treated with temporary, artificial or autologous skinreplacement. Agents that promote cell adhesion could speed healing.Blastocyst attachment to the endometrium of the uterine wall isessential for pregnancy and is a cause of infertility. Agents thatpromote blastocyst attachment could prevent infertility. Unfortunately,signaling pathways that regulate cell adhesion are largely unknown.

We have identified a pathway, the activin receptor type II (ActRII)signaling pathway, that regulates cell adhesion. Blocking ActRIIsignaling alters cellular morphology and increases cell detachment. Celldetachment correlates with an increase in the expression of ADAM-15, adisintegrin which cleaves integrin molecules (5) and cadherin (6). Inthis context, it has recently been demonstrated that the expression ofADAM-15 is strongly correlated with the metastatic potential ofprostate, breast (7) and pancreatic cancers (8) and is highlyup-regulated in aggressive prostate cancer (6). Furthermore, ADAM-15 hasbeen shown to be involved in cell migration and invasion (9, 10). Signaltransduction components of the activin signaling pathway are highlydown-regulated in prostate cancer (11, 12). In vitro, inhibition ofADAM15 expression in PC-3 cells decreases cell migration and adhesion tospecific extracellular matrix proteins, and is accompanied by areduction in the cleavage of N-cadherin by ADAM 15 at the cell surface(6). In patients with bone metastasis from prostate cancer, circulatinglevels of activin A are significantly higher (13). These findingsindicate that ActRII mediates cell adhesion (and viability) via theregulation of ADAM-15 expression and function.

Modulation of ActRII signaling and associated disintegrins can be usedto identify drugs that enhance ActRII signaling and promote cellattachment, or inhibit the expression and/or function of disintegrinsassociated with promoting cell detachment. Accordingly, it is desirableto provide a method, kit, and apparatus for utilizing ActRII signalingand associated disintegrins to identify compounds and conditions capableof modulating adhesion characteristics of cell.

SUMMARY OF THE INVENTION

The foregoing needs are met, to a great extent, by the presentinvention, wherein in one respect methods and kits for identifyingagents that inhibit cell detachment and increase cell attachment, and tothe use of such agents to develop methods of preventing, treating oralleviating and/or the symptoms of cancer, and other diseases andconditions is provided. Some examples of such diseases and conditionsare described in [0003] and [0005]. More specifically, the presentinvention is directed to the identification of agents that could be usedto treat cancer, or other conditions and diseases described in [0003]and [0005] where cell detachment or migration is detrimental, or wherecell adhesion is beneficial. The methods may include detecting, eitherdirectly or indirectly, agents that increase ActRII signaling, and/ordecrease disintegrin or metalloprotease expression and/or function, andprevent cell detachment and cell invasion. Conversely, methods mayinclude detecting, either directly or indirectly, agents that decreaseActRII signaling, and/or increase disintegrin or metalloproteaseexpression and/or function, and increase cell attachment and cellinvasion.

An embodiment of the present invention pertains to a method ofidentifying an agent to modify cell adhesion. In this method, cells froman animal are adhered to a matrix, cell detachment from a matrix isinduced by suppressing ActRII signaling, the agent is introduced, and achange in cell detachment as a result of introducing the agent ismeasured.

Another embodiment of the present invention relates to a kit to identifyan agent to modify cell adhesion. The kit includes a cell growth media,a container having a surface matrix for cell adhesion, and a celldetachment solution having a concentration of an ActRII signalingsuppressor sufficient to induce cell detachment.

Yet another embodiment of the present invention pertains to acomposition to induce cell detachment comprising a concentration of anActRII signaling suppressor sufficient to induce cell detachment.

There has thus been outlined, rather broadly, certain embodiments of theinvention in order that the detailed description thereof herein may bebetter understood, and in order that the present contribution to the artmay be better appreciated. There are, of course, additional embodimentsof the invention that will be described below and which will form thesubject matter of the claims appended hereto.

In this respect, before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notlimited in its application to the details of construction and to thearrangements of the components set forth in the following description orillustrated in the drawings.

The invention is capable of embodiments in addition to those describedand of being practiced and carried out in various ways. Also, it is tobe understood that the phraseology and terminology employed herein, aswell as the abstract, are for the purpose of description and should notbe regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the ActRII signaling pathway linked to the expressionand function of the disintegrin ADAM15.

FIG. 2 illustrates that ActRII blocking antibody suppression of ActRIIsignaling promotes morphological changes, increased detachment anddecreased viability of prostate cancer cells.

FIG. 3 illustrates that anti-ActRII oligonucleotide (antisense-P)suppression of ActRII signaling promotes increased ADAM15 expression,increased detachment and decreased viability of prostate cancer cells.

FIG. 4 illustrates that suppressing ActRII signaling promotesmorphological changes, increased detachment and decreased viability ofprostate cancer cells.

FIG. 5 illustrates antisense oligonucleotide suppression of ActRII alongwith ADAM-15 suppression to prevent cell detachment.

DEFINITIONS

The present invention is described herein using several definitions, asset forth below and throughout the application.

As used in this specification and the claims, the singular forms “a,”“an,” and “the” include plural forms unless the content clearly dictatesotherwise.

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the term “including” has the same meaning as the term“comprising.”

As used herein, the term “cancer cell line” or “cancer cells” may beused interchangeably and refers to cells isolated from a tumor,metastasis or abnormal growth derived from an animal or human. Thesecells typically, but not always, grow rapidly in culture whensupplemented with appropriate growth factors, often fetal animal serum.This term also refers to normal cells transformed into cells thatdisplay typical features of cancer cells, i.e. they divide in cellculture under trophic support and/or form tumors when administered toanimals.

Cancer cell lines may include, but are not limited to, human prostatecancer cells (PC-3, LNCaP, DU-145), human mammary epithelial cells (e.g.MCF-7, MCF-10A, MDA-MB-438, MDA-231, MDA-468, T47D, SkBr3), humanneuronal cells (M17, SHSY5Y, H4, U87), human acute myeloid leukemiacells (THP-1), human bone cancer cells (Saos-2 cells), human melanomacells (721), human glioblastoma cells (A172), human head and neckcarcinoma cells (A253), human skin epithelial cells (A431), human lungcarcinoma epithelial cells (A-549), human peripheral blood mononuclearcell lymphoma (BCP-1), human pancreatic adenocarcinoma cells (BxPC3),human squamous cell carcinoma (Cal-27), human CML acute phase T cellleukemia cells (CML T1), human CML blast crisis Ph+ CML cells (EM2),human CML blast crisis Ph+ CML cells (EM3), human metastatic lymph nodemelanoma cells (FM3), human lung cancer cells (H1299), human hybridomacells (HB54), human fibroblasts (HCA2), human kidney embryonicepithelial cell (HEK-293), human cervical cancer epithelial cell (HeLa),human myeloblast blood cells (HL-60), human mammary epithelial cells(HMEC), human colon epithelial adenocarcinoma cells (HT-29), humanumbilical cord vein endothelial cells (HUVEC), human T-cell-leukemiawhite blood cells (Jurkat), human lymphoblastoid EBV immortalized Bcells (JY cells), human lymphoblastoid CML blast crisis cells (K562cells), human lymphoblastoid erythroleukemia cells (Ku812), humanlymphoblastoid CML cells (KCL22), human lymphoblastoid AML cells (KG1),human lymphoblastoid CML cells (KY01), human melanoma cells (Ma-Mel 1,2, 3 through 48), human WBC myeloid metaplasic AML cells (MONO-MAC 6),human T cell leukemia (Peer), human osteosarcoma cells (Saos-2), human Tcell leukemia/B cell line hybridoma (T2), human colorectalcarcinoma/lung metastasis epithelium cells (T84), human colorectaladenocarcinoma cells (HCT-15, HT-29), human monocyte AML cells (THP1),human glioblastoma-astrocytoma epithelial cells (U373), humanglioblastoma-astrocytoma epithelial-like cells (U87), human leukemicmonocytic lymphoma cells (U937), human lymphoblastoid cells (WT-49),human B-cell EBV transformed cells (YAR), human breast adenocarcinomacells (NCl/ADR-RES, MDA-MB-231), human CNS glioblastoma cells (SF-268),human ovary adenocarcinoma cells (SK-OV-3), human lung carcinoma cells(NCII-1460), human lung adenocarcinoma cells (A549), human livercarcinoma cells (Hep3B), human uterine sarcoma-drug sensitive cells(MES-SA), human uterine sarcoma-drug resistant cells (MES-SA/DX5), humanskin primary melanoma cells (WM39), ape-kidney fibroblast cells (COS-7),African green monkey kidney epithelial cells (Vero cells), murinebrain/cerebral cortex endothelial cells (bEnd.3), murine embryonicmesenchymal cells (C3H-10T112), murine T cell leukemia ECACC cells(EL4), murine embryonic fibroblasts (NIH-3T3), murine embryoniccalvarial cells (MC3T3), murine hepatoma epithelial cell (Hepal cic7),murine adenocarcinoma cells (MC-38), murine epithelial cells (MTD-1A),murine endothelial cells (MyEnd), murine renal carcinoma cells (RenCa),murine melanoma cells (X63), murine lymphoma cells (YAC-1), murine Tcell tumor cells (RMA/RMAS), murine breast adenocarcinoma cells (4T1),murine mammary normal epithelial cells (NmuMG), rat glioblastoma cells(9L), rat neuroblastoma cells (B35), canine mammary tumor cells (CMT),canine osteosarcoma cells (D17), caninehistiocytosismonocyte/macrophages (DH82), rat pheochromocytoma cells(PC-12), rat pituitary tumor (GH3), canine kidney epithelial cells (MDCKII), murine B lymphoma B cells (lymphocyte A20), murine bone marrowstromal cells (ALC), murine melanoma cells (B16), murine colorectalcarcinoma cells (CT26), baby hamster kidney fibroblasts (BHK-21), Asiantiger mosquito larval tissue (C6/36), insect-ovary cells (Sf-9), Chinesehamster ovary cells (CHO), onyvax prostate cancer cells (OPCN, OPCT),tobacco cells (BY-2), zebrafish cells (ZF4 and AB9), Madin-Darby CanineKidney (MDCK) epithelial cells, Xenopus kidney epithelial cells (A6).

As used herein, the term “primary cells” refers to cells isolated fromtissues of animals.

Primary cells include, but are not limited to, keratinizing epithelialcells, wet stratified barrier epithelial cells, exocrine secretoryepithelial cells, holinone secreting cells, metabolism and storagecells, barrier function cells of lung, gut, exocrine glands andurogenital tract, kidney cells, epithelial cells lining closed internalbody cavities, ciliated cells, extracellular matrix secretion cells,blood and immune system cells, cells of the nervous system, autonomicneuron cells, sense organ and peripheral neuron supporting cells,central nervous system neurons and glial cells, lens cells, pigmentcells, germ cells, nurse cells, interstitial cells, stem cells and othercells (see Appendix 1 for list of cells).

A “cell array” refers to a substrate comprising a plurality of cancer orprimary cell lines.

The present methods and kits may utilize cell arrays to test agents forcell adhesion, cell invasion and cell viability.

The term “oligonucleotide” or “siRNA” refers to a nucleic acid sequenceor fragments or portions thereof, which may be single or doublestranded, and represents the sense or antisense strand. Aoligonucleotide may include DNA or RNA, and may be of natural orsynthetic origin. For example, a oligonucleotide or siRNA may includecDNA or mRNA. Oligonucleotides may include nucleic acid that has beenamplified (e.g., using polymerase chain reaction). The oligonucleotidemay contain phosphorothioate bonds.

The term “oligonucleotide” is understood to be a molecule that has asequence of bases on a backbone comprised mainly of identical monomerunits at defined intervals. The bases are arranged on the backbone insuch a way that they can enter into a bond with a nucleic acid having asequence of bases that are complementary to the bases of theoligonucleotide. The most common oligonucleotides have a backbone ofsugar phosphate units. Oligonucleotides of the method which function asantisense oligonucleotides are generally at least about 10-15nucleotides long and more preferably at least about 15 to 25 nucleotideslong, although shorter or longer oligonucleotides may be used in themethod. The exact size will depend on many factors, which in turn dependon the ultimate function or use of the oligonucleotide. Anoligonucleotide (e.g., antisense sequence) that is specific for a targetnucleic acid will “hybridize” to the target nucleic acid under suitableconditions. As used herein, “hybridization” or “hybridizing” refers tothe process by which an oligonucleotide single strand anneals with acomplementary strand through base pairing under defined hybridizationconditions. Oligonucleotides used as antisense oligonucleotides forspecifically inactivating gene transcription or translation are capableof specifically hybridizing to the target gene.

The present methods and kits may utilize oligonucleotides. The term“antisense” and “sense” are complimentary to “antisense oligonucleotide”and “sense oligonucleotide”, respectively, and refers to anoligonucleotide that hybridizes to a target nucleic acid and is capableof specifically hybridizing to the target nucleic acid. Antisense orsense may be fully complementary to a target nucleic acid sequence orpartially complementary. The level of complementarity will depend onmany factors based, in general, on the function of the antisense orsense oligonucleotide. An antisense oligonucleotide can be used, forexample to prevent gene transcription and translation for the formationof proteins. A sense oligonucleotide does not prevent gene transcriptionand translation for the foil-nation of proteins. Antisense and senseoligonucleotides can be labeled or unlabeled, or modified in any of anumber of ways well known in the art. An antisense and senseoligonucleotides may specifically hybridize to a target nucleic acid.

As used herein, a “target nucleic acid” refers to a nucleic acidmolecule containing a sequence that has at least partial complementaritywith a target DNA/RNA sequence. A target DNA/RNA may specificallyhybridize to a target nucleic acid.

As used herein, an “inhibitor” refers to a molecule that blocks ordecreases the function, phosphorylation or translocation of a protein orthe binding of a protein to another.

An “amino acid sequence” refers to a polypeptide or protein sequence.

The term “blocking antibody” refers to an antibody that blocks normalsignaling by that protein, usually by binding to a particular amino acidsequence of that protein.

As used herein, the term “agent,” which may be used interchangeably withthe terms “chemical”, “inhibitor” or “drug,” refers to any compound thatcan be applied to cells.

As used herein, the term “chemical library” means a group of distinctdrugs and drug classes maintained as a group that are testedindividually or in combination for their ability to affect a cellularchange, e.g. cell adhesion.

As used herein, the term “assay” or “assaying” means qualitative orquantitative analysis or testing.

As used herein, the term “cell viability” refers to whether a cell isliving or dead. Cell viability measurements may be used to evaluate thedeath or life of cells. Cell viability tests can be used to determinethe effectiveness of agents to promote life, or induce death.

As used herein, the term “cell invasion” and “migrate” refers to theability of a cell to move from its starting position to a new place,under the influence of chemotaxic agents or not.

As used herein, the term “cell attachment” and “cell adhesion” refers tocell binding to a substrate, including but not limited to plastic andother substrates referred to in [0049] and the like.

As used herein, the term “ADAM-15 expression and function” refers to theconcentration of ADAM-15 protein, and the activity of that protein toact as a metalloproteinase to cleave ECM proteins.

As used herein, the term “ActRI, Smad-2 and Smad-4 expression andphosphorylation” refers to the concentrations of ActRI, Smad-2 andSmad-4, and whether ActRI, Smad-2 and Smad-4 are phosphorylated.

As used herein, the term “ActRII binding to ALKs” refers to the bindingof one member of the ActRII family of proteins to another member of theALK family of proteins.

As used herein, the term “Smad-2:Smad-4 coupling” refers to the bindingof Smad2 to Smad-4 as a complex.

DETAILED DESCRIPTION

In some embodiments, the methods include: (a) using cancer cell linesand primary cells; (b) attaching cells to a matrix; (c) inducingdetachment of cancer cells or primary cells by blocking the ActRIIsignaling pathway using oligonucleotides, oligonucleotides withphosphorothioate bonds (antisense-P phospho-oligonucleotides), siRNA,blocking antibodies, or inhibitors of protein function, binding ortranslocation; (d) using agents from chemical libraries or other agents;(e) measuring cell attachment; (f) measuring cell invasion; (g)measuring cell viability; (h) measuring the expression and activity ofdisintegrins, (i) measuring ActRII, activin receptor-like kinases (ALKs)1-7, Smad-2, Smad-3 and Smad-4 expression and their phosphorylation; (j)measuring ActRII binding to ALKs; (k) measuring Smad-2:Smad-4 couplingand translocation to the nucleus. In one method, cancer cell lines areinduced to detach from a culture or microwell plate and agents aretested to determine their ability to prevent detachment and cellinvasion.

In a preferred embodiment, the determination of the ability of the agentto inhibit cell detachment is made by treating cultured cancer cellswith antisense-P phosphooligonucleotides and then treating the cellswith chemical agents and measuring cell detachment, invasion, morphologyand viability.

In some embodiments, the methods may include using plastic cultureplates, or plastic culture plates that are coated with different bindingsubstrates, including but not limited to fibronectin, vitronectin,bovine serum albumin, gelatin, Matrigel, fibrous matrix proteins (suchas collagen I, collagen IV), fibrinogen, non-collagenous components(such as laminin molecules; GTFALRGDNGDNGQ—portion of the lamininalpha-chain), proteoglycans (such as chondroitin sulfate, dermatansulfate, heparin, heparan sulfate and keratan sulfate, syndecans,perlecan), non-sulfated glycosaminoglycan (such as hyaluronan),entactin, core proteins (such as lumican, keratocan, mimecan,fibromodulin, PRELP, osteoadherin and aggrecan), dystroglycanglycoprotein complex and Lutheran blood group glycoprotein. Plates mayconsist of one or more wells (e.g. 6-, 12-, 24-, 48-, 96-, 384-well) forhigh throughput screening of agents.

In some embodiments, the methods may include using any cancer cell linethat can adhere to the surface of the plate.

In some embodiments, the methods may include using any primary cellsfrom animals, or human cells from biopsies, that can adhere to thesurface of the plate.

In some embodiments, the methods may include using, but are not limitedto oligonucleotides, oligonucleotides with phosphorothioate bonds, RNAinterference (RNAi) or inhibitors for decreasing the expression orfunction of the activin signaling pathway including ActRIIA and B,activin receptor-like kinases (ALKs 1-7), Smad-2, Smad-3, Smad-4,activins (A, B, C, D and E). In another embodiment, the methods mayinclude using blocking antibodies against members of the activinsignaling pathway including ActRII, ActRI, Smad-2, Smad-4, activins (A,B, C, D and E), for decreasing protein function. In another embodiment,the methods may include using oligonucleotides, oligonucleotides withphosphorothioate bonds, RNAi or inhibitors to decrease phosphorylationof activin signaling pathway members. In another embodiment, the methodsmay include using oligonucleotides, oligonucleotides withphosphorothioate bonds, RNAi or inhibitors to inhibit ActRIIA and/orActRIIB binding to ALKs 1-7. In another embodiment, the methods mayinclude using oligonucleotides, oligonucleotides with phosphorothioatebonds, RNAi or inhibitors to decrease Smad-2:Smad-4 translocation to thenucleus. In another embodiment, the methods may include natural cellularinhibitors of the signal transduction pathway of ActRII signaling suchas inhibin and follistatin.

The treatments may include single agents, combinations of agents or noagents. The treatments also may include solutions used to dissolve theagents.

Cell detachment and adhesion may be detected by any suitable method,which may include, but are not limited to, cell cytometry (e.g. trypanblue), fluorescent based cell detection assays (e.g. Calcein AM(InVitrogen, Inc.), and Mitotracker Red (InVitrogen, Inc.), luminescentbased detection assays (e.g. Cell-Titer glo (Promega, Inc.) andspectrophotometry based detection assays (e.g. crystal violet, MTS/MTTassays such as Promega CellTiter 96® AQueous Non-Radioactive CellProliferation Assay, Promega, Inc. and Chem icon Cell Adhesion Assays).

Cell invasion may be detected by any suitable method, which may include,but are not limited to, the scratch wound assay, cell invasion assaysusing fluorescent detection of cell invasion (e.g. activin, serum) (BDBioCoat matrigel invasion chambers, Fisher Scientific; EMD, Calbiochem;Chemicon International) with or without the use of a chemotaxic agent.

Cell viability may be detected by any suitable method, which mayinclude, but are not limited to, cell cytometry (e.g. trypan blue),fluorescent based cell detection assays (e.g. calcein AM (InVitrogen,Inc.), and Mitotracker Red (InVitrogen, Inc.)), luminescent baseddetection assays (e.g. Cell-Titer glo (Promega, Inc.)) andspectrophotometry based detection assays (e.g. crystal violet, MTS/MTTassays such as Promega CellTiter 96® AQueous Non-Radioactive CellProliferation Assay, Promega, Inc.)

Cell morphology may be assessed by capturing images using a microscopeand analyzing morphological changes using software such as MetaMorph(Molecular Devices).

The disclosed methods will determine which agents or combination ofagents prevent cell detachment, prevent cell invasion, promote cellattachment, promote cell invasion, increase viability, decreaseviability and alter morphology.

The disclosed methods may be utilized to identify agents that: 1)prevent metastasis or the other conditions/diseases such as thosedescribed in [0005]; 2) detach cells from a matrix; and 3) promote celladhesion to a matrix. In addition, the disclosed methods may be utilizedto study cell surface markers during cell detachment and how agentsaffect the expression and activity of those markers. Also contemplatedare kits for performing the disclosed methods. A kit may include one ormore reagents for determining, either directly or indirectly, whether anagent(s) can prevent cell detachment, prevent cell invasion, promotecell attachment, promote cell invasion and alter morphology/viability.

The present methods may be performed to induce cell detachment from amatrix and to identify agents that prevent cell detachment. Cells arecultured to attain 80% confluence as known in the art. Typically cellsare cultured under sterile conditions at 37° C., 5% CO2, in mediasupplemented with 1% penicillin-streptomycin, 2 mM glutamine, 0.4 mMsodium bicarbonate and 1-10% fetal bovine serum as determined by thecell type and as known in the art. Cells are then treated with antisenseoligonucleotides with phosphorothioate bonds or RNAi to a finalconcentration of 0.1-10 μM. Oligonucleotides can be added directly, oradded to media that has been preincubated with lipofectamine (4 ng/til;InVitrogen Corporation, Carlsbad, Calif.) for 5 min. at roomtemperature, and this mixture then incubated at room temperature for 20min. prior to addition to cells. Cell attachment, invasion and viabilityare significantly altered by suppressing ActRII signaling via thesetreatments. Agent(s) are then added to cells at concentrations rangingfrom picomolar to millimolar concentrations. Fluids that the agent(s)are dissolved in are added to separate wells at the same concentration.Other control wells contain no agent(s). After 0-5 days of treatment,cell attachment, invasion, morphology and viability are measured asdescribed in [0054], [0055] and [0056].

The ability of agents to prevent cell detachment is measured by thedifference in cell attachment between those cells treated with andwithout agents. Alternatively, the percentage change in cell attachmentby an agent can be measured by the level of cell attachment in thepresence of the agent and oligonucleotides or blocking antibody dividedby the difference between cells treated with and withoutoligonucleotides/or antibody alone as known in the art.

Cell detachment can be induced by, but is not limited to, the followingoligonucleotides:

SEQ ID NO 1 ActRIIA antisense-P:  5′-TCCAGTTCAGAGTCCCATTTC-3′SEQ ID NO 2 ActRIIA sense: 5'-GAAATGGGACTCTGAACTGGA-3' SEQ ID NO 3ActRIIB antisense-P: 5'-TCTCCCGTTCACTCTGCCAC-3' SEQ ID NO 4Act RUB sense: 5'-GTGGCAGAGTGAACGGGAGA-3' SEQ ID NO 5ADAM-15 Antisense-P: 5'-CGCACTCTTCCCTGGTAGCA-3' SEQ ID NO 6ADAM-15 Sense: 5'-TGCTACCAGGGAAGAGTGCG-3'

Cell detachment can be induced by, but is not limited to, the followingantibodies and inhibitors: Anti-human ActRIIB affinity-purified mousemonoclonal antibody (A0856; US Biological, Swampscott, Mass.);Anti-human mouse activin antibody (US Biologicals, MA); SB 431542(GlaxoSmithKline). Any oligonucleotide or antibody that decreasessignaling via the ActRII signaling pathway can be used to induce celldetachment.

The present methods may be performed to identify agents that promotecell attachment and prevent cell detachment from a matrix. Cellscultured as described in [0060] at 50% confluence are treated with andwithout agents and after 0-5 days cell attachment, invasion andviability are measured in any suitable manner. Specific examples ofsuitable methods of measuring cell attachment, invasion and viabilityinclude thus methods described in [0054], [0055] and [0056].

The present methods may be used for identifying agents that prevent celldetachment and promote cell adhesion. These agents may be used to treatany suitable metastasis and other diseases and conditions. Specificexamples of suitable metastasis and other diseases and conditionsinclude those described in [0005].

In a particular embodiment of the invention, a kit for identifying agentthat prevent ActRII-mediated cell detachment is provided. The kit mayinclude any suitable reagents for performing various assays capable ofidentifying agent that prevent ActRII-mediated cell detachment. In aparticular example, the kit includes:

-   -   1) sense and antisense-P oligonucleotides to ActRII    -   2) lipofectamine    -   3) 96-well plate (coated with a particular matrix for different        cell types). Plate would be either visible, fluorescent of        luminescent light compatible. Cells would be dependent upon each        researchers requirements as would the media that they would put        the lipofectamine/oligonucleotides into for treatment of cells.    -   4) PBS buffer (for washing wells)    -   5) cell detection reagents

In addition, depending upon the cell number detection method, the kitmay include various additional reagents for performing particular assayssuch as, for example, fluorescent detection of cell number, luminescentdetection of cell number, spectrophotometric detection of cell number,and the like. For fluorescent detection of cell number, the kit mayinclude reagents for calcein AM (e.g., fluorescent compatible 96-wellplate). For luminescent detection of cell number, the kit may includereagents for CellTiter-Glo® (e.g., luminescent compatible 96-wellplate). For spectrophotometric detection of cell number, the kit mayinclude reagents for crystal violet assay such as:

1) PBS buffer (for washing wells)

2) crystal violet solution (0.5%; staining cells)

3) 33% (v/v) acetic acid (digesting cells)

4) (visible light compatible 96-well plate)

To perform the spectrophotometric detection of cell number: Media isremoved from cells cultured in 96-well plates, the cells washed withD-PBS (Gibco, Carlsbad, Calif., USA), and 50 μL of crystal violet addedto each well at room temperature for 10 min. Following this, 200 μL of33% (v/v) acetic acid is added to the wells, the plate shaken for 2 min.and then read at 570 nm on a plate reading spectrophotometer.

Methods and Results Example 1 ActRII Oligonucleotide-Induced Detachmentof Cancer Cells in a Microwell Format for the High Throughput Screeningof Small Molecules to Inhibit Cell Detachment

Prostate cancer cell lines (e.g. PC-3 cells) are cultured in 96-wellopaque plates (luminescence compatible) at 37° C. in 100 μL of F-12Nutrient Mixture (Ham; Gibco, InVitrogen Corporation, Carlsbad, Calif.)supplemented with 1% penicillin-streptomycin (P/S; Gibco, InVitrogen), 2mM glutamine (InVitrogen), 0.4 mM sodium bicarbonate (Sigma, St. Louis,Mo.), and 5% fetal bovine serum (FBS, #26400-036; Gibco, InVitrogen).When cells reach 80% confluence (at least 24 h after plating to allowsuitable attachment), cells are treated every day for 1-3 days with:

-   -   1) medium+0.4 μM ActRIIB sense-P oligonucleotide (SEQ ID NO 4);        or    -   2) medium+0.4 μM ActRIIB antisense-P oligonucleotide (SEQ ID NO        3); or    -   3) medium+0.4 μM ActRIIB antisense-P oligonucleotide (SEQ ID NO        3)+small molecule (0-10 μM).    -   4) medium+0.4 μM ActRIIB sense-P oligonucleotide (SEQ ID NO        4)+small molecule (0-10 μM).

The oligomers with phosphorothioate bonds (sense and antisense-P;Integrated DNA Technology, Coralville, Iowa) are added to media that hasbeen preincubated with lipofectamine (4 ng/μl; InVitrogen Corporation,Carlsbad, Calif.) for 5 min. at room temperature. This mixture isincubated at room temperature for 20 min. prior to addition to cells.Replicates=3-6. As a modification to the above protocol, antisenseoligonucleotides can be replaced with a specific binding antibody toActRil such as anti-human ActRIIB affinity-purified mouse monoclonalantibody (A0856; US Biological, Swampscott, Mass.).

The number of viable cells attached to the plate is determined using theCellTiter-Glo® Luminescent Cell Viability Assay (Promega). Briefly, theCellTiter-Glo® Reagent is added directly to the wells of the plate (100μL per well for 96-well plates) and the plate mixed on an orbital shakerfor 2 min. Following this, the plate is incubated at room temperaturefor 10 min. and the luminescence recorded (typical integration time of0.25-1 second per well) on a plate-reading luminometer. Note: Controlwells are prepared containing medium without cells to obtain a value forbackground luminescence, and is subtracted from the values obtained forcells treated in 1, 2 and 3 above.

The % of cell detachment rescued by the small molecules is determined asthe luminescence of (3−2)/(1−2)×100.

Example 2 Antisense Oligonucleotide Suppression of ActRII Along withADAM-15 Suppression Prevents Cell Detachment

To test that cell detachment induced by suppressing ActRII signaling wasmediated via the metalloprotease ADAM-15, we treated PC-3 prostaticcancer cells with antisense-P against ActRII and then treated thesecells with antisense-P against ADAM-15. A significant decrease in cellattachment was detected after 3 days of treatment with antisense-Pagainst ActRII, and this was reversed by treatment with antisense-Pagainst ADAM-15 (p<0.01, n=5). As shown in FIG. 5, neither sense againstActRII nor together with sense against ADAM-15 altered cell attachment.These results illustrate that cell detachment induced by suppressingActRII signaling is mediated via the metalloprotease ADAM-15, andfurther indicate that antisense-P against ADAM-15 as a chemical that canprevent prostate cancer cell detachment.

Androgen-insensitive PC3 cells derived from a grade IV human prostateadenocarcinoma of epithelial origin (examples described herein) weremaintained at 37° C. in F-12 Nutrient Mixture (Ham; Gibco, InVitrogenCorporation, Carlsbad, Calif.) supplemented with 1%penicillin-streptomycin (P/S; Gibco, InVitrogen), 2 mM glutamine(InVitrogen), 0.4 mM sodium bicarbonate (Sigma, St. Louis, Mo.), and 5%fetal bovine serum (FBS, #26400-036; Gibco, InVitrogen). For theexperiment, PC-3 cells were plated in 6 well plates with 10% serum at2×10⁵ cells/well for 24 h, after which cells treated every day for 3days with:

-   -   1) media containing 10% serum+lipofectamine+sense-P        oligonucleotide to ActRIIB (sActRIIB) (SEQ ID NO 4);    -   2) media containing 10% serum+lipofectamine+antisense-P        oligonucleotide to ActRIIB (aActRIIB) (SEQ ID NO 3);    -   3) media containing 10% serum+lipofectamine+sense-P        oligonucleotide to ActRIIB (sActRIIB) (SEQ ID NO 4)+sense-P        oligonucleotides to ADAM-15 (SEQ ID NO 6)    -   4) media containing 10% serum+lipofectamine+aActRIIB (SEQ ID NO        3)+antisense-P oligonucleotides to ADAM-15 (SEQ ID NO 5).

The oligomers with phosphorothioate bonds (Sense and antisense-P;Integrated DNA Technology, Coralville, Iowa) were added to media (240μl) that had been preincubated with lipofectamine (4 ng/μl; InVitrogenCorporation, Carlsbad, Calif.) for 5 min. at room temperature. Thismixture was then incubated at room temperature for 20 min. prior toaddition to cells. Antisense-P was used at a final concentration of 0.4μM. The number of viable PC-3 cells attached to the plate was measuredafter trypsinization followed by cell counting using the trypan bluestaining assay at the end of 72 h. Results are presented as cellattachment (% of sense control; mean±SEM, n=5). Statistical differencesare denoted by an * p<0.01 as shown in FIG. 5. In addition to themethods and results presented herein, methods and results described in“Simon D, Vadakkadath Meethal S, Wilson A C, et al. Activin receptorsignaling regulates prostatic epithelial cell adhesion and viability.Neoplasia 11:365-376” the disclosure of which is incorporated herein inits entirety.

The many features and advantages of the invention are apparent from thedetailed specification, and thus, it is intended by the appended claimsto cover all such features and advantages of the invention which fallwithin the true spirit and scope of the invention. Further, sincenumerous modifications and variations will readily occur to thoseskilled in the art, it is not desired to limit the invention to theexact construction and operation illustrated and described, andaccordingly, all suitable modifications and equivalents may be resortedto, falling within the scope of the invention.

REFERENCES

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APPENDIX 1

Keratinizing epithelial cells such as epidermal keratinocyte(differentiating epidermal cell), epidermal basal cell (stem cell),keratinocyte of fingernails and toenails, nail bed basal cell (stemcell), medullary hair shaft cell, cortical hair shaft cell, cuticularhair shaft cell, cuticular hair root sheath cell, hair root sheath cellof Huxley's layer, hair root sheath cell of Henle's layer, external hairroot sheath cell, hair matrix cell (stem cell).

Wet stratified barrier epithelial cells such as surface epithelial cellof stratified squamous epithelium of cornea, tongue, oral cavity,esophagus, anal canal, distal urethra and vagina, basal cell (stem cell)of epithelia of cornea, tongue, oral cavity, esophagus, anal canal,distal urethra and vagina, urinary epithelium cell (lining urinarybladder and urinary ducts).

Exocrine secretory epithelial cells such as salivary gland mucous cell(polysaccharide-rich secretion), salivary gland serous cell(glycoprotein enzyme-rich secretion), von Ebner's gland cell in tongue(washes taste buds), mammary gland cell (milk secretion), lacrimal glandcell (tear secretion), ceruminous gland cell in ear (wax secretion),eccrine sweat gland dark cell (glycoprotein secretion), eccrine sweatgland clear cell (small molecule secretion), apocrine sweat gland cell(odoriferous secretion, sex-hormone sensitive), gland of Moll cell ineyelid (specialized sweat gland), sebaceous gland cell (lipid-rich sebumsecretion), Bowman's gland cell in nose (washes olfactory epithelium),Brunner's gland cell in duodenum (enzymes and alkaline mucus), seminalvesicle cell (secretes seminal fluid components, including fructose forswimming sperm), prostate gland cell (secretes seminal fluidcomponents), bulbourethral gland cell (mucus secretion), Bartholin'sgland cell (vaginal lubricant secretion), gland of Littre cell (mucussecretion), uterus endometrium cell (carbohydrate secretion), isolatedgoblet cell of respiratory and digestive tracts (mucus secretion),stomach lining mucous cell (mucus secretion), gastric gland zymogeniccell (pepsinogen secretion), gastric gland oxyntic cell (hydrochloricacid secretion), pancreatic acinar cell (bicarbonate and digestiveenzyme secretion), Paneth cell of small intestine (lysozyme secretion),yype II pneumocyte of lung (surfactant secretion), Clara cell of lung.

Hormone secreting cells such as anterior pituitary cells, somatotropes,lactotropes, thyrotropes, gonadotropes, corticotropes, intermediatepituitary cell, secreting melanocyte-stimulating hormone, magnocellularneurosecretory cells, cells secreting oxytocin, secreting vasopressin,gut and respiratory tract cells, secreting serotonin, secretingendorphin, secreting somatostatin, secreting gastrin, secretingsecretin, secreting cholecystokinin, secreting insulin, secretingglucagon, secreting bombesin, thyroid gland cells, thyroid epithelialcell, parafollicular cell, parathyroid gland cells, parathyroid chiefcell, oxyphil cell, adrenal gland cells, chromaffin cells, secretingsteroid hormones (mineralcorticoids and gluco corticoids), leydig cellof testes secreting testosterone, theca interna cell of ovarian folliclesecreting estrogen, corpus luteum cell of ruptured ovarian folliclesecreting progesterone, granulosa lutein cells, theca lutein cells,juxtaglomerular cell (renin secretion), macula densa cell of kidney,peripolar cell of kidney, mesangial cell of kidney.

Metabolism and storage cells such as hepatocyte (liver cell), white fatcell, brown fat cell, liver lipocyte.

Barrier function cells of lung, gut, exocrine glands and urogenitaltract.

Kidney cells such as kidney glomerulus parietal cell, kidney glomeruluspodocyte, kidney proximal tubule brush border cell, Loop of Henle thinsegment cell, kidney distal tubule cell, kidney collecting duct cell.

Epithelial cells lining closed internal body cavities such as bloodvessel and lymphatic vascular endothelial fenestrated cell, blood vesseland lymphatic vascular endothelial continuous cell, blood vessel andlymphatic vascular endothelial splenic cell, synovial cell (lining jointcavities, hyaluronic acid secretion), serosal cell (lining peritoneal,pleural, and pericardial cavities), squamous cell (lining perilymphaticspace of ear), squamous cell (lining endolymphatic space of ear),columnar cell of endolymphatic sac with microvilli (lining endolymphaticspace of ear), columnar cell of endolymphatic sac without microvilli(lining endolymphatic space of ear), dark cell (lining endolymphaticspace of ear), vestibular membrane cell (lining endolymphatic space ofear), stria vascularis basal cell (lining endolymphatic space of ear),stria vascularis marginal cell (lining endolymphatic space of ear), cellof Claudius (lining endolymphatic space of ear), cell of Boettcher(lining endolymphatic space of ear), choroid plexus cell (cerebrospinalfluid secretion), pia-arachnoid squamous cell, pigmented ciliaryepithelium cell of eye, nonpigmented ciliary epithelium cell of eye,corneal endothelial cell.

Ciliated cells with propulsive function such as respiratory tractciliated cell, oviduct ciliated cell (in female), uterine endometrialciliated cell (in female), rete testis ciliated cell (in male), ductulusefferens ciliated cell (in male), ciliated ependymal cell of centralnervous system (lining brain cavities).

Extracellular matrix secretion cells such as ameloblast epithelial cell(tooth enamel secretion), planum semilunatum epithelial cell ofvestibular apparatus of ear (proteoglycan secretion), organ of Cortiinterdental epithelial cell (secreting tectorial membrane covering haircells), loose connective tissue fibroblasts, corneal fibroblasts, tendonfibroblasts, bone marrow reticular tissue fibroblasts, othernonepithelial fibroblasts such as pericyte, nucleus pulposus cell ofintervertebral disc, cementoblast/cementocyte (tooth root bonelikecementum secretion), odontoblast/odontocyte (tooth dentin secretion),hyaline cartilage chondrocyte, fibrocartilage chondrocyte, elasticcartilage chondrocyte, osteoblast/osteocyte, osteoprogenitor cell (stemcell of osteoblasts), hyalocyte of vitreous body of eye, stellate cellof perilymphatic space of ear contractile cells such as skeletal musclecells, red skeletal muscle cell (slow), white skeletal muscle cell(fast), intermediate skeletal muscle cell, nuclear bag cell of musclespindle, nuclear chain cell of muscle spindle, satellite cell (stemcell), heart muscle cells such as ordinary heart muscle cell, nodalheart muscle cell, purkinje fiber cell, smooth muscle cell (varioustypes), myoepithelial cell of iris, myoepithelial cell of exocrineglands.

Blood and immune system cells such as erythrocyte (red blood cell),megakaryocyte (platelet precursor), monocyte, connective tissuemacrophage (various types), epidermal Langerhans cell, osteoclast (inbone), dendritic cell (in lymphoid tissues), microglial cell (in centralnervous system), neutrophil granulocyte, eosinophil granulocyte,basophil granulocyte, mast cell, helper T cell, suppressor T cell,cytotoxic T cell, natural Killer T cell, B cell, natural killer cell,reticulocytes, stem cells and committed progenitors for the blood andimmune system (various types).

Cells of the nervous system such as sensory transducer cells whichinclude auditory inner hair cell of organ of Corti, auditory outer haircell of organ of Corti, basal cell of olfactory epithelium (stem cellfor olfactory neurons), cold-sensitive primary sensory neurons,heat-sensitive primary sensory neurons, merkel cell of epidermis (touchsensor), olfactory receptor neuron, pain-sensitive primary sensoryneurons (various types), photoreceptor cells of retina in eye(Photoreceptor rod cells, photoreceptor blue-sensitive cone cell of eye,photoreceptor green-sensitive cone cell of eye, photoreceptorred-sensitive cone cell of eye), proprioceptive primary sensory neurons(various types), touch-sensitive primary sensory neurons (varioustypes), type I carotid body cell (blood pH sensor), type II carotid bodycell (blood pH sensor), type I hair cell of vestibular apparatus of ear(acceleration and gravity), type II hair cell of vestibular apparatus ofear (acceleration and gravity), type I taste bud cell.

Autonomic neuron cells such as cholinergic neural cell (various types),adrenergic neural cell (various types), peptidergic neural cell (varioustypes).

Sense organ and peripheral neuron supporting cells such as inner pillarcell of organ of Corti, outer pillar cell of organ of Corti, innerphalangeal cell of organ of Corti, outer phalangeal cell of organ ofCorti, border cell of organ of Corti, Hensen cell of organ of Corti,vestibular apparatus supporting cell, type I taste bud supporting cell,olfactory epithelium supporting cell, Schwann cell, satellite cell(encapsulating peripheral nerve cell bodies), enteric glial cell.

Central nervous system neurons and glial cells such as astrocyte(various types), neuron cells (large variety of types, still poorlyclassified), oligodendrocyte, spindle neuron.

Lens cells such as anterior lens epithelial cell, crystallin-containinglens fiber cell.

Pigment cells such as melanocyte, retinal pigmented epithelial cell.

Germ cells such as oogonium/oocyte, spermatid, spermatocyte,spermatogonium cell

(stem cell for spermatocyte), spermatozoon.

Nurse cells such as ovarian follicle cell, sertoli cell (in testis),thymus epithelial cell.

Interstitial cells like interstitial kidney cells.

Stem cells such as embryonic stem cells, mesenchymal stem cells,subcutaneous preadipocytes, visceral preadipocytes, osteoclastprecursors, neural progenitors, bone marrow mononuclear cells, cordblood mononuclear cells, bone marrow progenitors, cord bloodprogenitors, cord blood erythroid progenitors, fetal liver progenitors,bone marrow stromal cells.

Other cells such as type I pneumocyte (lining air space of lung),pancreatic duct cell (centroacinar cell), nonstriated duct cell (ofsweat gland, salivary gland, mammary gland, etc.), principal cell,intercalated cell, duct cell (of seminal vesicle, prostate gland, etc.),intestinal brush border cell (with microvilli), exocrine gland striatedduct cell, gall bladder epithelial cell, ductulus efferens nonciliatedcell, epididymal principal cell, epididymal basal cell.

1. A method of identifying an agent to modify cell adhesion comprisingthe steps of: adhering cells from an animal to a matrix; inducing celldetachment from a matrix by suppressing ActRII signaling; introducingthe agent; and measuring a change in cell detachment as a result ofintroducing the agent.
 2. The method according to claim 1, furthercomprising: subjecting the cells to antisense ActRII DNA to suppressActRII signaling.
 3. The method according to claim 2, wherein theantisense ActRII DNA has the sequence of SEQ ID
 3. 4. The methodaccording to claim 1, wherein the modifications to cell adhesion includeone or more of preventing cell detachment, preventing cell invasion,promoting cell adhesion, and promoting cell invasion.
 5. The methodaccording to claim 4, further comprising: measuring changes in thesignaling of ActRII pathway members and disintegrin family members. 6.The method according to claim 5, further comprising: measuringexpression, phosphorylation, function and translocation of ActRIIsignaling pathway members and disintegrin family members.
 7. The methodaccording to claim 1, further comprising: suppressing ActRII signalingvia subjecting the cells to one or more of antisense oligonucleotides;antisense oligonucleotides with phosphorothioate bonds; RNAi; blockingantibodies; and other chemical or natural inhibitors of proteinfunction.
 8. The method according to claim 7, further comprising:suppressing ActRII signaling via one or more of: (a) inhibiting activinreceptor type HA or type IIB expression; (b) inhibiting activin receptortype IIA or type IIB phosphorylation; (c) inhibiting activin receptortype IIA and/or type IIB binding to ALKs 1-7; (d) blocking ALKs 1-7phosphorylation; (e) inhibiting ALK-4, -5 and -7 expression; (f)blocking ALK phosphorylation of Smad-2 and/or Smad-3; (g) inhibitingSmad-2 and/or Smad-3 expression; (h) blocking phosphorylated Smad-2and/or Smad-3 from binding to Smad-4; and (i) blocking Smad2:Smad4translocation to the nucleus.
 9. The method according to claim 1,wherein the cells include one or more of cancer cells, transformedcells, and primary cells.
 10. A kit to identify an agent to modify celladhesion, the kit comprising: a cell growth media; a container having asurface matrix for cell adhesion; and a cell detachment solution havinga concentration of an ActRII signaling suppressor sufficient to inducecell detachment.
 11. The kit according to claim 10, further comprising:sense and antisense-P oligonucleotides to ActRII.
 12. The kit accordingto claim 10, further comprising: lipofectamine.
 13. The kit according toclaim 10, further comprising: a 96-well mirotiter plate for growing thecells.
 14. The kit according to claim 10, further comprising: a celldetection reagent.
 15. The kit according to claim 14, furthercomprising: a fluorescent cell detection reagent.
 16. The kit accordingto claim 14, further comprising: a luminescent cell detection reagent.17. A composition to induce cell detachment comprising a concentrationof an ActRII signaling suppressor sufficient to induce cell detachment.18. The composition according to claim 17, wherein the ActRII signalingsuppressor includes ActRIIB antisense having a sequence identical to SEQID NO
 3. 19. The composition according to claim 17, wherein the ActRIIsignaling suppressor includes ActRIIA antisense having a sequenceidentical to SEQ ID NO 1.